This application seeks funding for studies aimed at dissecting the down-regulation of adaptive immune responses directed against coagulation factors that are introduced by protein or gene replacement therapy into individuals with the X-linked bleeding disorder hemophilia. These potentially detrimental responses not only result in the formation of inhibitory antibodies against factor VIII (deficient in hemophilia A) or factor IX (F.IX, deficient in hemophilia B), they also include the generation of cytotoxic T lymphocytes, which can eliminate cells that are modified upon gene transfer. Whilst both sets of responses are dependent on antigen specific helper CD4+ T cells, adaptive immune responses to F.IX are suppressed by naive or antigen specific regulatory T cells (Treg, Tr1, NKT). Nonetheless, we are only beginning to understand the implications of immune regulation in the treatment of hemophilia, as we need to dissect the genetic and mechanistic underpinnings of these regulatory processes. The experiments proposed in this Competing Renewal application are designed to test the general hypothesis that several regulatory T cell subsets cooperate in the prevention of a primary response or mitigate an existing response to F.IX introduced via AAV-based gene transfer or as a protein. The proposed experiments are grouped into the following Specific Aims: i) To optimize the in vitro generation of F.IX specific Treg. ii) To test the hypothesis that in vivo antibody and CD8+ T cell responses directed against F.IX can be suppressed by induction by Treg. iii) To test the hypothesis that conventional Treg, IL-10 producing Treg and NKT cells regulate immune responses directed against AAV-hF.IX. Together these experiments should clarify the basic mechanisms, which govern inhibitor and T cell responses to coagulation factors and therapeutic proteins. The results could directly be used for the design of therapeutic strategies that can be applied treatment of hemophilia.